Chitin (or a derivative thereof) is a polysaccharide composition prepared from the shells of arthropods, including crustaceans or insects. The material is biocompatible and naturally resorbed by the body, and has been previously used for sustained drug release, bone induction and hemostasis (Chandy and Sharma, Biomat. Art. Cells & Immob. Biotech. 19:745-760 (1991); Klokkevold, P. et al., J. Oral Maxillofac. Sur. 50:41-45 (1992)).
Surgical adhesives and tissue sealants have been used for sealing internal and external wounds, such as in bones and skin, to reduce blood loss and maintain hemostasis. Biologically-derived tissue sealants typically contain blood clotting factors and other blood proteins. One type of tissue sealant, referred to as fibrin sealant or fibrin glue, is a gel similar to a natural blood clot which is prepared from plasma. The precise components used to produce a specific fibrin sealant are a function of the particular plasma fraction which is used as a starting material; however, typically fibrin glue contains a mixture of proteins which disadvantageously include thrombin and traces of albumin, fibronectin and plasminogen, which upon contact are destructive of certain classes of plasma proteins, e.g., Factor IX. In addition, bioadhesives have proven unsuccessful in part because in many cases commercially available preparations of fibrin glue and tissue sealants are too dense to allow cell migration into and through the material to permit satisfactory wound healing. This limits their effectiveness in many in vivo uses for which a long-felt need remained until the discovery of the present chitin hydrogel as an effective delivery system
a. Plasma Proteins
Plasma proteins are any protein found within the plasma of a patient, or which is found in the plasma of a normal individual or animal, but which is absent or deficient in the patient (e.g, hemophilias). Particularly relevant to the present invention are plasma protein members of the blood clotting cascade, including those clotting factors which are thrombin sensitive; that is, those blood clotting factor molecules which have been shown to include one or more binding sites for thrombin. All coagulation proteins (e.g., Factors VIII, IX, VIIa, Protein C) have short circulating half-lives. Upon exposure to circulating thrombin, the thrombin-sensitive plasma proteins, such as Factor IX and possibly Factor VIII are soon inactivated.
The chitin hydrogel of the present invention, however, provides an effective system for the delivery of intact plasma proteins, including thrombin-sensitive plasma proteins. Plasma proteins include, but are not limited to, the following: albumin; immunoglobulins, including immunoglobulin A, M and G; fibrinogen; coagulation factors, including Factors II, VII, VIII, IX, X and XIII; plasminogen; protein C; protein S; plasma proteinase inhibitors, including antithrombin III, .alpha.1-antitrypsin, .alpha.2-macroglobulin, and C1 esterase inhibitor; .alpha.1-acid glycoprotein; ceruloplasmin; haptoglobin; transferrin; complement components C1 through C9; C4b binding protein; interalpha-trypsin inhibitor; apolipoproteins, including A-1, A-11, B, C and E; fibronectin and angiostatin.
b. Growth Factors
When a tissue is injured, polypeptide growth factors, which exhibit an array of biological activities, are released into the wound where they play a crucial role in healing (see, e.g., Hormonal Proteins and Peptides (Li, C. H., ed.) Volume 7, Academic Press, Inc., New York, N.Y. pp. 231-277 (1979) and Brunt et al., Biotechnology 6:25-30 (1988)). These activities include recruiting cells, such as leukocytes and fibroblasts, into the injured area, and inducing cell proliferation and differentiation. Growth factors that may participate in wound healing include, but are not limited to: platelet-derived growth factors (PDGFs); insulin-binding growth factor-1 (IGF-1); insulin-binding growth factor-2 (IGF-2); epidermal growth factor (EGF); transforming growth factor-.alpha. (TGF-.alpha.); transforming growth factor-.beta. (TGF-.beta.); platelet factor 4 (PF-4); and heparin binding growth factors one and two (HBGF-1 and HBGF-2, respectively).
PDGFs are stored in the alpha granules of circulating platelets and are released at wound sites during blood clotting (see, e.g., Lynch et al., J. Clin. Invest. 84:640-646 (1989)). PDGFs include: PDGF; platelet derived angiogenesis factor (PDAF); TGF-.beta.; and PF4, which is a chemoattractant for neutrophils (Knighton et al., in Growth Factors and Other Aspects of Wound Healing: Biological and Clinical Implications, Alan R. Liss, Inc., New York, N.Y., pp. 319-329 (1988)). PDGF is a mitogen, chemoattractant and a stimulator of protein synthesis in cells of mesenchymal origin, including fibroblasts and smooth muscle cells. PDGF is also a nonmitogenic chemoattractant for endothelial cells (see, for example, Adelmann-Grill et al., Eur. J. Cell Biol. 51:322-326 (1990)).
IGF-1 acts in combination with PDGF to promote mitogenesis and protein synthesis in mesenchymal cells in culture. Application of either PDGF or IGF-1 alone to skin wounds does not enhance healing, but application of both factors together appears to promote connective tissue and epithelial tissue growth (Lynch et al., Proc. Natl. Acad. Sci. 76:1279-1283 (1987)).
TGF-.beta. is a chemoattractant for macrophages and monocytes. Depending upon the presence or absence of other growth factors, TGF-.beta. may stimulate or inhibit the growth of many cell types.
Other growth factors, such as EGF, TGF-.alpha., the HBGFs and osteogenin are also important in wound healing. Topical application of EGF accelerates the rate of healing of partial thickness wounds in humans (Schultz et al., Science 235:350-352 (1987)). Osteogenin, which has been purified from demineralized bone, appears to promote bone growth (see, e.g., Luyten et al., J. Biol. Chem. 264:13377 (1989)). In addition, platelet-derived wound healing formula, a platelet extract which is in the form of a salve or ointment for topical application, has been described (see, e.g., Knighton et al., Ann. Surg. 204:322-330 (1986)).
The heparin binding growth factors (HBGFs), including the fibroblast growth factors (FGFs), which include acidic HBGF (aHBGF also known as HBFG-1 or FGF-1) and basic HBGF (bHBGF also known as HBGF-2 or FGF-2), are potent mitogens for cells of mesodermal and neuroectodermal lineages, including endothelial cells (see, e.g., Burgess et al., Ann. Rev. Biochem. 58:575-606 (1989)). In addition, HBGF-1 is chemotactic for endothelial cells and astroglial cells. Both HBGF-1 and HBGF-2 bind to heparin, which protects them from proteolytic degradation. The array of biological activities exhibited by the HBGFs suggests that they play an important role in wound healing.
Basic fibroblast growth factor (FGF-2) is a potent stimulator of angiogenesis and the migration and proliferation of fibroblasts (see, for example, Gospodarowicz et al., Mol. Cell. Endocinol. 46:187-204 (1986) and Gospodarowicz et al., Endo. Rev. 8:95-114 (1985)). Acidic fibroblast growth factor (FGF-1) has been shown to be a potent angiogenic factor for endothelial cells (Burgess et al., supra, 1989). Other FGF's may be chemotactic for fibroblasts. Growth factors are, therefore, potentially useful for specifically promoting wound healing and tissue repair.
However, to date, the art has provided inadequate means for applying a growth factor to a wound to achieve a prolonged contact between the wound and the growth factor. This problem has been overcome by the chitin hydrogel of the present invention.
c. Bone Wounds and Their Repair
The sequence of bone induction was first described by Urist et al. using demineralized cortical bone matrix (Clin. Orthop. Rel. Res. 71:271 (1970) and Proc. Natl. Acad. Sci. USA 70:3511 (1973)). Implanted subcutaneously in allogeneic recipients, demineralized cortical bone matrix releases factors which act as local mitogens to stimulate the proliferation of mesenchymal cells (Rath et al., Nature (Lond.) 278:855 (1979)). New bone formation occurs between 12 and 18 days postimplantation. Ossicle development replete with hematopoietic marrow lineage occurred by day 21 (Reddi, A., In Extracellular Matrix Biochemistry (Piez et al., ed.) Elsevier, New York, N.Y., pp. 375-412 (1984)).
Demineralized bone matrix (DBM) is a source of osteoinductive proteins known as bone morphogenetic proteins (BMP), and growth factors which modulate the proliferation of progenitor bone cells (see, e.g., Hauschka et al., J. Biol. Chem. 261:12665-12674 (1986) and Canalis et al., J. Clin. Invest. 81:277-281 (1988)). Eight BMPs have now been identified and are abbreviated BMP-1 through BMP-8. BMP-3 and BMP-7 are also known as osteogenin and osteogenic protein-1 (OP-1), respectively.
Unfortunately, DBM materials have little clinical use unless combined with particulate marrow autografts. There is a limit to the quantity of DBM that can be surgically placed into a recipient's bone to produce a therapeutic effect. In addition, resorption has been reported to be at least 49% (Toriumi et al., Arch. Otolaryngo. Head Neck Surg. 116:676-680 (1990)).
DBM powder and osteogenin may be washed away by tissue fluids before their osteoinductive potential is expressed. In addition, seepage of tissue fluids into DBM-packed bone cavities or soft-tissue collapse into the wound bed are two factors that may significantly affect the osteoinductive properties of DBM and osteogenin. Soft-tissue collapse into the wound bed may likewise inhibit the proper migration of osteocompetent stem cells into the wound bed. Moreover, DBM in powder form is difficult to use.
Purified BMPs have osteoinductive effects in animals when delivered by a variety of means including fibrin glue (Hattori, T., Nippon. Seikeigeka. Gakkai. Zasshi. 64:824-834 (1990); Kawamura et al., Clin. Orthop. Rel. Res. 235:302-310 (1988); Schlag et al., Clin. Orthop. Rel. Res. 227:269-285 (1988) and Schwarz et al., Clin. Orthop. Rel. Res. 238:282-287 (1989)) and whole blood clots (Wang et al., J. Cell. Biochem. 15F:Q20 Abstract (1990)). However, Schwarz et al. (supra.) demonstrated neither a clear positive or negative effect of a matrix on ectopic osteoinduction or BMP-dependent osteoregeneration. Kawamura et al. (supra.) found a synergistic effect when partially purified BMP in a biomatrix was tested in an ectopic non-bony site. Consequently, the prior art presents an inconsistent and confusing picture of the applicability of delivery of an osteogenic supplement to a patient from a tissue sealant.
d. Vascular Prostheses
Artificial vascular prostheses, frequently made of dacron or polytetrafluoroethylene (PTFE), are used to replace diseased blood vessels in humans and other animals. To maximize patency rates and minimize the thrombogenicity of vascular prostheses, various techniques have been used including seeding of nonautologous endothelial cells onto the prothesis. Various substrates which adhere both to the vascular graft and endothelial cells have been investigated as an intermediate substrate to increase endothelial cell seeding. However, the use of nonautologous cells for the seeding the surface of the substrate raises the possibility of tissue rejection. See e.g., Schrenk et al., Thorac. Cardiovasc. Surg. 35:6-10 (1986). In addition, a confluent endothelium usually requires months to established, if it can be established at all. The delay results in a high rate of failure due to occlusion of the vascular prosthesis (see, e.g., Zilla et al., Surgery 105.515-522 (1989). This problem has been overcome by the chitin hydrogel of the present invention.
e. Angiogenesis
Angiogenesis is the induction of new blood vessels. Certain growth factors such as HBGF-1 and HBGF-2 are angiogenic. However, their in vivo administration when attached to: collagen sponges (Thompson et al., Science 241:1349-1352 (1988)); beads (Hayek et al., Biochem. Biophys. Res. Commun. 147:876-880 (1987)); solid PTFE fibers coated with collagen arranged in a sponge-like structure (Thompson et al., Proc. Natl. Acad. Sci. USA 86:7928-7932 (1989)); or by infusion (Puumala et al., Brain Res. 534:283-286 (1990)) resulted in the generation of random, disorganized blood vessels. Until the discovery of the present invention, these growth factors had not been used successfully to direct the growth of new blood vessels at a given site in vivo.
f. Site-Directed, Localized Drug Delivery
An efficacious, site-directed, drug delivery system is greatly needed in several areas of medicine. For example, localized drug delivery is needed in the treatment of local infections, such as in periodontitis, where the systemic administration of antimicrobial agents is ineffective. The problem after systemic administration usually lies in the low concentration of the antimicrobial agent which can be achieved at the target site. To raise the local concentration a systemic dose increase may be effective, but it also may produce toxicity, microbial resistance and drug incompatibility.
To circumvent some of these problems, several alternative methods have been devised, but none are ideal. For example, collagen and/or fibrinogen dispersed in an aqueous medium as an amorphous flowable mass, and a proteinaceous matrix composition which is capable of stable placement, have also been shown to locally deliver drugs (Luck et al., U.S. Reissue Pat. No. 33,375; Luck et al., U.S. Pat. No. 4,978,332). Fibrin sealant have been used to deliver a variety of antibiotics, but only at relatively low concentrations and for relatively short periods of time ranging from a few hours to a few days (Kram et al., J. Surg. Res. 50:175-178 (1991)). Most of the antibiotics have been in freely water soluble forms and have been added during the preparation of the tissue sealant. However, their delivery is hampered by the limitations of the fibrin delivery system, which are overcome by the chitin hydrogel of the present invention.
The incorporation of tetracycline hydrochloride tetracycline hydrochloride (TET HCl) and other freely water soluble forms of antibiotic has often interfered with polymerization during the formation of an antibiotic-supplemented fibrin matrix (Schlag et al., Biomaterials 4:29-32 (1983)). Thus, the amount and concentration of the TET HCl that could be formulated with the hydrogel may be antibiotic-concentration dependent.
g. Controlled Drug Release
For some clinical applications controlled, localized drug release is desirable. Although, some drugs, especially antibiotics, have been incorporated into and been released from biomatrices, little or no control over the duration of the drug release has been possible. This is, at least partially, a reflection of the relatively short life of the drug-supplemented biomatrix. Therefore, there remains a long-felt need in the art to provide a means for extended, localized drug release, as are new techniques for the incorporation and extended release of other supplements from a biocompatible delivery system. This need is satisfied by the chitin hydrogel of the present invention.
Local implantation of antibiotics in a matrix has become popular in the treatment of wounds, such as open fractures or acute and chronic osteomyelitis. Several substances have been employed as the delivery vehicle, with polymethylmethacrylate (PMMA) being the most commonly utilized vehicle (Buchholz, H. W. et al., J. Bone Joint Surgery (British) 63:342-353 (1981); Buchholz, H. W. et al., Clin. Orthop. 190:96-108 (1984); Christian, E. P. et al., J. Bone Joint Surg. (American) 71:994-1004 (1989); Majid, S. A. et al., Acta Orthop. Scand. 56:265-268 (1985)). PMMA-antibiotic, which is surgically implanted after debriment of wound, is capable of significantly increasing local tissue levels of antibiotics while decreasing dead space. However, the major disadvantage of PMMA therapy is it is non-resorbable. If left in the wound after the antibiotic has eluted, it can act as a foreign body and additional surgery is required for its removal.
For example, acute osteomyelitis is a rapidly progressing infection of bone whereas the chronic form of osteomyelitis results from a long-standing infection of the bone (Waldvogel, F A, "Acute Osteomyelitis," In: Orthopaedic Infection, ed. by D. Schlossberg, New York, Springer-Verlag, 1988, p1-8). Acute osteomyelitis can be successfully treated with antibiotics provided the disease is diagnosed early, while for the chronic form, successful treatment requires debriment of the wound and administration of antibiotics. Antibiotics, an adjunct to thorough debridement, usually are administered systemically. However, conventional antibiotic systemic therapy results in the release of a therapeutically ineffective levels of antibiotics at the site of infection (Dash, A. and Suryanarayanan, R., Pharmaceut. Res. 9:993-1002 (1992)). Systemic treatment can also result in serious toxicity. Furthermore, the cost of large amounts of antibiotics used for systemic treatment may restrict therapy.
One solution to overcome the negative effects of systemic antibiotic therapy for osteomyelitis is local antibiotic delivery. Local antibiotic delivery can reduce systemic side effects by using a fraction of the systemic antibiotic dose to combat the infection. Local deposition of antibiotics has become increasingly popular in the treatment of osteomyelitis (Waldvogel (1988), supra), and several substances have been used as antibiotic delivery vehicles, however, none provide the advantages of the chitin hydrogel of the present invention.
h. The Disclosed Preparations Provide Life-Saving Emergency Treatment for Trauma Wounds
Despite continued advances in trauma care, a significant percentage of the population, both military and civilian, suffer fatal or severe hemorrhage every year. An alarming number of fatalities are preventable since the occur in the presence of those who could achieve life-saving control of their wounds given adequate tools and training. The availability of the herein-disclosed chitin hydrogel satisfies the long-felt need for a advanced, easy-to-use, field-ready delivery system which can be effectively combined with the advantages of a hemostatic preparation.
The disclosed technology would also be available for the treatment of massed casualties in disaster situation, when the availability of the easy-to-use, self-contained hydrogel preparations disclosed below will permit local medical personnel and disaster relief workers to provide the injured with temporary treatment until definitive care becomes available. Moreover, the disclosed chitin hydrogel preparations will permit self-treatment in disaster victims, until medical assistance can be provided.